Cancer results from an accumulation of genetic and epigenetic changes in cancer genes, including tumor suppressor genes;these changes cause normal somatic cells to evolve into cancer cells. In Knudson's two-hit paradigm, loss of tumor suppressor gene function occurs in two steps. The second of those steps can occur by multiple mechanisms including chromosome loss, recombination, insertion, deletion, point mutation, or epigenetic modification. In order to find vertebrate genes involved in those steps, which serve to maintain genome stability, we have used the zebrafish (Danio rerio) to perform a screen for genomic instability mutants that models this second hit. This screen used the mosaic eye assay, in which pale cells appear in a black background of retinal pigmented epithelial (RPE) cells that are heterozygous for a recessive pigment mutation, goldenb1. Twelve ENU- induced genomic instability (gin), or somatic mutator mutations were isolated in this screen at a rate suggesting the potential existence of 200 such genes. Most of the gin mutations showed weak dominance in heterozygotes, and all showed a stronger phenotype in homozygotes. The strongest mutant, gin-10, showed a striking (~10-fold) phenotypic enhancement over homozygous phenotype when inherited maternally in trans with other gin mutations (the "gin-10 interacting group");gin-10 carriers show a 9.6-fold increase in susceptibility to spontaneous cancer. These findings represent first steps in our long-term goal of developing the zebrafish as a vertebrate model for finding genes involved in the control of genomic stability in vertebrates. Now that we have shown that the zebrafish can be used to find somatic mutators that are susceptible to cancer, we propose to elucidate the nature of the striking interactions between the genes comprising the gin-10 interacting group, and to better-characterize the associated tumor susceptibility. This will be accomplished in three Specific Aims. For Specific Aim 1, we will clone members of the gin-10 interacting group using positional cloning for gin-10 and gin-12, and using a sensitized insertional screen with a novel "gene breaking" To12 transposon-based vector. Specific Aim 2 is to characterize the mechanism of genomic instability using microsatellite, interphase FISH analysis, and exon sequencing of the golden gene in golden RPE cells from mosaic eyes and by similar analysis of p53 in gin-10-associated tumors. Specific Aim 3 is to determine the tumor susceptibility of the strongly mosaic gin-10/gin-12 trans-heterozygotes. The proposed studies will contribute to our understanding of gene interactions involved in somatic loss of gene function, and to clarify how they contribute to cancer susceptibility. It is our hope that such understanding will lead to novel ways to prevent or treat human cancer. PUBLIC HEALTH RELEVANCE: Cancer is a disease in which normal cells accumulate changes in their DNA as they evolve into cancer cells. We now know that the genes controlling the stability of our DNA play a key role in cancer. We are using the unique power of the zebrafish as a vertebrate model system to find and study genes that are important in genetic stability and cancer, in the hope that our increased understanding will lead to measures to treat and prevent human cancer.